Compositions and methods for promoting nerve regeneration

ABSTRACT

FK506 and geldanamycin promote nerve regeneration by a common mechanism that involves the binding of these compounds to polypeptide components of steroid receptor complexes other than the steroid hormone binding portion of the complex (FKBP52 and hsp90, respectively). These and other agents cause hsp90 dissociation from steroid receptor complexes or block association of hsp90 with steroid receptor complexes.

This application is a divisional application of Ser. No. 08/956,691,filed Oct. 24, 1997, now issued as U.S. Pat. No. 5,968,921.

BACKGROUND OF THE INVENTION

Following traumatic or mechanically induced axonal degeneration in theperipheral nervous system, axonal regeneration ensues, resulting infunctional recovery. However, the rate of axonal elongation (3-4 mm/day)is slow. Consequently, recovery is measured in weeks or months,depending upon the distance between the site of injury and the targettissue. Therapies that speed regeneration over long distances would behighly beneficial to patients and would significantly reduce health carecosts.

The immunosuppressant drug FK506 (USAN tacrolimus; Prograf®) speedsfunctional recovery and axonal regeneration in the rat in adose-dependent manner following a sciatic nerve crush lesion (Gold etal., J. Neurosci. 15:7505-7516, 1995; Gold et al., Restor. Neurol.Neurosci. 6:287-296, 1994). FK506 was shown to stimulate neuriticoutgrowth in a rat pheochromocytoma cell line in aconcentration-dependent manner (Lyons et al., Proc. Natl. Acad. Sci. USA91:3191-3195, 1994).

Systemic administration of two synthetic FK506 analogs that bind FKBP-12but that do not inhibit calcineurin activity (and which are notimmunosuppressants) increases the size of myelinated fibers (Steiner etal., Nature Medicine 3:1-8, 1997; Steiner et al., Proc. Natl. Acad. Sci.USA 94:2019-2024, 1997). U.S. Pat. No. 5,654,332 (Armistead et al.)discusses immunosuppressive FK506 analogs that bind FKBP12 and that aresaid to stimulate neurite outgrowth in the presence of NGF. It wasstated that the neurotrophic activity of these FKBP12 binding compounds“is directly related to their affinity for FKBP12 and their ability toinhibit FKBP12 rotomase activity” (id. at col. 7, lines 47-50).

It has been reported that androgens and estrogens stimulate facial nerveregeneration in hamsters (Jones, “Androgenic enhancement of motor neuronregeneration,” In: Luine and Harding, eds., Hormonal Restructuring ofthe Adult Brain, Ann. N.Y. Acad. Sci. 85:141-164, 1994; Tanzer andJones, Exp. Neurol. 146:25814 264, 1997).

SUMMARY OF THE INVENTION

I have discovered that geldanamycin and FK506 stimulate nerveregeneration via a common mechanism. Both compounds bind to polypeptidecomponents of steroid receptor complexes, hsp90 and FKBP52,respectively. These and other compounds that cause hsp90 dissociationfrom steroid receptor complexes or that block association of hsp90 withsteroid receptor complexes stimulate nerve cell growth and promote nerveregeneration. Such coupounds can act directly by binding to hsp90 (as inthe case of geldanamycin) or indirectly by binding to anotherpolypeptide in the steroid receptor complex (as in the case of FK506binding of FKBP52).

According to one aspect of the invention, pharmaceutical compositionsare provided that include a nerve growth stimulating amount of anon-FKBP12-binding agent that binds to a polypeptide component of asteroid receptor complex other than the ligand (i.e., steroid hormone)binding portion thereof (such polypeptide components including, but notlimited to, hsp90 or FKBP52) and a pharmaceutically acceptableexcipient. Without limitation to any particular mechanism of action,binding of such agents to the polypeptide component likely causes hsp90dissociation from the complex or prevents hsp90 association with thecomplex. Nerve growth promoting agents according to the inventioninclude, but are not limited to non-FKBP12-binding FK506 analogs,benzoquinone ansamycins (e.g., geldanamycin and derivatives thereof),peptides that comprise a sequence of a selected polypeptide component ofthe complex at a site of interaction between the selected component andanother component of the complex, antibodies that bind a polypeptidecomponent of the steroid receptor complex, and combinations thereof.

According to another aspect of the invention, such pharmaceuticalcompositions include other active ingredients, including, but notlimited to, neurotrophic factor other than the nerve growth promotingagent (e.g., NGF, IGF-1, aFGF, bFGF, PDGF, BDNF, CNTF, GDNF, NT-3, NT4/5, or mixtures thereof), and a steroid ligand of the steroid receptorcomplex (e.g., estrogen and dexamethasone, as in the Examples below).

According to another aspect of the invention, a transection (severing ofthe nerve) of a periphal nerve or a spinal cord injury of a mammal istreated by methods that include administering a nerve growth stimulatingamount of a non-FKBP12-binding nerve growth promoting agent to themammal and grafting to the peripheral nerve or spinal cord an allograftor an artificial nerve graft. In the case of a transected peripheralnerve or spinal cord, the space between the transected ends of theperipheral nerve or spinal cord is preferably filled with a materialsuch as collagen, methyl cellulose, etc., or a cell suspension thatpromotes nerve cell growth, such as Schwann cells and olfactory andsheathing cells. The nerve growth promoting agent can be includedtogether with with such filling materials.

According to another aspect of the invention, pharmaceuticalcompositions are provided that include a nerve growth stimulating amountof a non-FKBP12-binding FK506 analog that binds to FKBP52 and apharmaceutically suitable excipient.

According to another aspect of the invention, pharmaceuticalcompositions are provided that include a nerve growth stimulating amountof an agent that binds to hsp90 and causes hsp90 dissociation from asteroid receptor complex or prevents hsp90 association with the complexand a pharmaceutically suitable excipient.

According to another aspect of the invention, methods of stimulatingnerve cell growth in a mammal are provided that include administering apharmaceutical composition as described herein.

According to another aspect of the invention, methods are provided forstimulating growth of a nerve cell that include contacting the nervecell with a non- FKBP12-binding agent that binds to a polypeptidecomponent of a steroid receptor complex other than the steroid hormonebinding portion thereof and causes hsp90 dissociation from the complexor blocks association of hsp90 with the complex.

According to another aspect of the invention, methods of identifyingcompounds that stimulate nerve cell growth are provided that include thesteps of assaying test compounds for binding to a component of a steroidreceptor complex other than the steroid hormone binding portion thereof,and assaying the binding compounds for stimulation of nerve cell growth.In addition, the binding compounds can be assayed for activity indissociation of hsp90 from the complex or blocking association of hsp90with the complex.

The foregoing and various features and advantages of the invention willbecome more apparent from the following detailed description andaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structures of FK506 (left) and a representative FK506analog, V-10,367 (right). The bracketed portion of FK506 represents thecalcineurin- binding domain, which is absent in V-10,367.

FIGS. 2-8 are histograms showing the stimulation of growth of SH-SY5Ycells by geldanamycin and FK506 in the presence of NGF (10 ng/mL) 168hours after treatment.

FIG. 2: control cells (untreated).

FIG. 3: NGF only (10 ng/mL).

FIG. 4: geldanamycin (1 nM)+NGF (10 ng/mL).

FIG. 5: geldanamycin (10 nM)+NGF (10 ng/mL).

FIG. 6: FK506 (10 nM)+NGF (10 ng/mL).

FIG. 7: geldanamycin (1 nM)+FK506 (10 nM)+NGF (10 ng/mL).

FIG. 8: geldanamycin (10 nM)+FK506 (10 nM)+NGF (10 ng/mL).

FIGS. 9-15 are histograms showing the stimulation of growth of SH-SY5Ycells by geldanamycin and FK506 in the presence of NGF (10 ng/mL) 168hours after treatment.

FIG. 9: control cells (untreated).

FIG. 10: NGF only (10 ng/mL).

FIG. 11: FK506 (1 nM)+NGF (10 ng/mL).

FIG. 12: FK506 (10 nM)+NGF (10 ng/mL).

FIG. 13: geldanamycin (0.1 nM)+NGF (10 ng/mL).

FIG. 14: geldanamycin (0.1 nM)+FK506 (1 nM)+NGF (10 ng/mL).

FIG. 15: geldanamycin (0.1 nM)+FK506 (10 nM)+NGF (10 ng/mL).

DETAILED DESCRIPTION

Members of the steroid/thyroid receptor family, including steroidreceptors such as the glucocorticoid and progesterone receptors, act asligand-inducible enhancers of specific gene expression.

Upon translation, steroid receptors are assembled into multiproteincomplexes. Steroid receptors exist in two states that are in dynamicequilibrium in the cell. An initial hormone receptor complex includesthe steroid receptor, hsp90, hsp70, and at least two co- chaperones, p60and Hip (p48). This initial complex is localized in the cytosol and doesnot bind DNA or enhance specific gene transcription. The initial complexis in equilibrium with a metastable, nearly mature complex that lackshsp90, p60, and Hip but includes p23 and one of the three largeimmunophilins, FKBP52 (also called hsp56), FKBPS4, or CyP40, and undersome circumstances, hsp 70 (Smith et al., Mol. Cell. Biol. 15:6804-6812,1995; Dittmar et al., J. Biol. Chem. 271:12833-12839, 1996). The nearlymature complex is competent to bind hormone; upon hormone binding, thereceptor is released as an active transcription factor (Smith et al.,Mol. Cell. Biol. 15:6804-6812, 1995; Dittmar et al., J. Biol. Chem.271:12833-12839, 1996). Steroid receptor complexes are constantlydissociating and reforming under physiological conditions.

Hsp90-mediated conformational maturation is required for nuclear hormonereceptors to acquire or maintain a state competent to bind hormone(Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995; Dittmar et al., J.Biol. Chem. 271:12833-12839, 1996). Geldanamycin, a benzoquinoneansamycin antibiotic, binds in a pharmacologically specific manner tohsp90 (Whitesell et al., Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994)and prevents association of the p23 component of the heterocomplexassembly system with hsp90 (Johnson and Toft, Mol. Endocrinol.9:670-678, 1995). Geldanamycin permits dissociation of a steroidreceptor complex, permitting receptors to be transformed (i.e.,dissociated from hsp90), but blocks reassembly of the hormone-responsive form of the complex, thereby preventing hormone activationand ultimately resulting in the degradation of the hormone receptor.Geldanamycin blocks assembly of the progesterone receptor (PR) complex(Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995) and of theglucocorticoid receptor (GR) complex (Czar et al., Biochem.36:7776-7785, 1997) at an intermediate stage of assembly where thehormone binding domain is not properly folded and therefore cannot bindsteroid. Geldanamycin also is known to act on estrogen and androgenhormone receptors (Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995;Nair et al., Cell Stress and Chaperones 1:237-250, 1996). Transformationof CR and PR as measured either by 9S to 4S conversion or by acquisitionof DNA-binding activity is correlated with dissociation of steroidreceptors from hsp90 (see, e.g., Meshinchi et al., J. Biol. Chem.265:4863-4870, 1990; Kost et al., Mol. Cell. Biol. 9:3829-3838, 1989).

In addition to steroid receptors, other “substrate proteins” for hsp90include v-erbA, dioxin receptor, sim, myoD1, E12, heat shock factor,tumor promoter-specific binding protein, hepatitis B reversetranscriptase, p53 tumor suppressor mutant, various protein kinases(e.g., the tyrosine kinases v-src, c-src, v-fps, v-yes, v-fes, v-frg,c-frg, lck, Weel kinase, and sevenless PTK), heme-regulated eIF-2α,eEF-2 kinase, casein kinase II, v-raf, c-raf, Gag-Mil, MEK, PI-4 kinase,actin, tubulin, centrin, proteasome, and G_(βγ)complex (reviewed inPratt and Toft, Endocrine Rev. 18:306-360, 1997).

FKBP52 is a member of the FK506-binding class of immunophilins. Bindingof FK506 to GR-associated FKBP52 caused increased nuclear translocationof GR in response to dexamethasone and potentiation of GR-mediated geneexpression (Sanchez and Ning, METHODS: A Companion to Meth. Enzymol.9:188-200, 1996). Dexamethasone- induced GR-specific gene expression isalso potentiated by cyclosporin A (CsA) (Renoir et al., Proc. Natl.Acad. Sci. USA 92:4977-4981, 1995), rapamycin (Ning and Sanchez, J.Biol. Chem. 268:6073-6076, 1993), and nonimmunosuppressive analogs ofFK506 (e.g., 15-o- desmethyl FK520) or of CsA (e.g., CsH and SDZ220384)(Sanchez and Ning, METHODS: A Companion to Meth. Enzymol. 9:188-200,1996).

CyP40, rather than FKBP52, is the target for binding of CsA and itsanalogs (Sanchez and Ning, METHODS: A Companion to Meth. Enzymol.9:188-200, 1996). FKBP52 and CyP40 bind directly to hsp90, and CyP40competes for FKBP52 binding to hsp90 and vice versa. The immunophilinsbind hsp90 in a mutually exclusive fashion, leading to the formation ofseparate CyP40-hsp90 and FKBP52-hsp90 complexes (Ratajczak and Carrello,J. Biol. Chem. 271:2961-2965, 1996). Immunophilins such as FKBP52 andCyP40 and non-immunophilin proteins such as PP5, p60, and Mas70p, haveone or more tetratricopeptide repeat (TPR) domains (Ratajczak et al., J.Biol. Chem. 268:13187-13192, 1993) that bind to the TPR-binding domainof hsp90. The number of TPR domains in a protein appears to correlatewith its hsp90-binding affinity. Regions bordering the TPR domain alsoparticipate in binding, e.g., the acidic domain at the N-terminal end ofbovine CyP40 (residues 185-225) and FKBP52 (residues 232-271) and thecalmodulin binding region at the C-terminus of bovine CyP40 (Ratajczakand Carrello, J. Biol. Chem. 271:2961-2965, 1996). Binding of bothFKBP52 and CyP40 to hsp90 is competed by a purified fragment of humanCyP40 comprising its three TPR domains and by a fragment of rat PP5comprising its four TPR domains (reviewed in Pratt and Toft, EndocrineRev. 18:306-360, 1997).

In addition to multiple TPR binding domains, FKBP52 contains a sequence(EDLTDDED SEQ ID NO:1 in rabbit) that is retained with conservativereplacements in human and mouse. This negatively charged sequence iselectrostatically complementary to the receptor nuclear localizationsignals (e.g., the NL1 sequence RKTKKKIK SEQ ID NO:2 of rat GR). Anantibody raised against the conserved negatively charged sequenceimpeded the dexamethasone- mediated shift of the GR into the nucleus(reviewed in Pratt and Toft, Endocrine Rev. 18:306-360, 1997). It hasalso been reported that antibodies directed against a conservednegatively-charged sequence of FKBP52 impede dexamethasone-mediatedcytophasmic-nuclear translocation of GR (Czar et al., Mol. Endocrinol.9:1549-1560, 1995).

The effects of FK506 and geldanamycin on nerve regeneration likelyresult from the binding of these compounds to components of steroidreceptor complexes, causing the dissociation of hsp90 from the steroidreceptor complex either directly (by binding to hsp90 or interferingwith the binding of hsp90 to the steroid receptor) or indirectly (bybinding to a polypeptide such as FKBP52 that itself binds to hsp90), or,alternatively, by preventing association of hsp90 with the steroidreceptor complex. However, interference with the ability of hsp90 tocomplex with and perform its chaperone function for other hsp90substrate proteins may also be responsible for or contribute to theobserved stimulation of nerve regeneration by FK506 and/or geldanamycin.

Definitions and Methods

“Nerve Growth Promoting Agent” (NGPA).

A “nerve growth promoting agent” or NGPA is defined as a substance thatbinds to a polypeptide component of a steroid receptor complex otherthan the steroid hormone binding portion thereof, such componentsincluding but not limited to hsp90 and FKBP52, and promotes nerveregeneration, without limitation to a particular mechanism of action.Preferably, the NGPA does not bind FKBP12 and is non-immunosuppressive.NGPAs include, but are not limited to, non-FKBP12-binding(“non-binding”) analogs of FK506; benzoquinone ansamycins, includinggeldanamycin, naturally occurring analogs of geldanamycin, including,but not limited to, herbimycin A and macbecin (DeBoer et al., J.Antibiot. (Tokyo) 23:442-447, 1970; Omura et al., J. Antibiot. (Tokyo)32:255-261, 1979; Ono et al., Gann. 73:938-944, 1992), and derivativesthereof; peptides including an amino acid sequence of a particularpolypeptide component of a steroid receptor complex at a site ofinteraction between that component and another component of the complex(such as the TPR domain), and antibodies that bind specifically topolypeptide components of steroid receptor complexes, (e.g., anti-hsp90,anti-FKBP52, etc.) and interfere with the interaction of the boundpolypeptide with another polypeptide in the steroid receptor complex.

“Steroid Receptor Complex” or “Steroid Hormone Receptor” and “Component”Thereof; “Transformation”; “Activation”.

The term “steroid receptor complex” is intended to encompass amultiprotein complex associated with any steroid receptor, including,but not limited to, the progesterone receptor, glucocorticoid receptor,estrogen receptor, androgen receptor, and mineralocorticoid receptor. Apolypeptide “component” is a polypeptide other than the steroid hormonebinding portion of the steroid receptor complex, and preferably otherthan steroid receptor (particularly the steroid hormone binding portionthereof), such as hsp90, FKBP52, etc., that is part of a steroidreceptor complex.

The term “transformation” refers to the conversion of the 9Snon-DNA-binding form of a steroid receptor complex to the 4S DNA-bindingform. The term “activation” refers to the conversion of a steroidreceptor from a form that does not bind steroid to a steroid-bindingform.

Assays for Identifying NGPAs.

There are a number of well-known methods for assaying compounds thatbind to hsp90, FKBP52, and other polypeptide components of a steroidreceptor complex that can be used as an initial screen for candidatecompounds that stimulate nerve regeneration. Compounds can subsequentlybe tested in vitro or in vivo for activity in stimulating nerveregeneration.

For example, one may assay for the binding of a test compound to apolypeptide that is a component of a steroid receptor complex. An assayfor binding to hsp90 is described, for example, by Whitesell et al.(Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994). Commercial hsp90(StressGen Biotechnologies, Victoria, BC) dissolved in 20 μg/mL of TNESVbuffer (50 mM Tris-HCl, pH 7.4/1% Nonidet P-40/2 mM EDTA/100 mM NaCl/imM orthovanadate/1 mM phenylmethylsulfonyl fluoride/20 μg leupeptin permL/20 μg of aprotinin per ml) and the test compound are incubated for 45min at 4° C. with geldanamycin immobilized on a conventional solidsupport, e.g., geldanamycin- coupled agarose beads (Whitesell et al.,Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994). The beads are thenwashed with TNESV buffer and bound hsp90 is eluted by heating inreducing loading buffer and can be analyzed by SDS/PAGE and silverstaining (Bio-Rad), for example. Alternatively, if the hsp90 is labeled,one can assay for bound label versus free label. Test compounds thatcompete with geldanamycin for binding to hsp90 inhibit the binding ofsolubilized hsp90 to the beads.

Similar assays can be performed to identify compounds that bind othersteroid receptor complex polypeptide components. Binding to FKBP52 canbe assayed using recombinant FKBP52 (Peattie et al., Proc. Natl. Acad.Sci. USA 89:10974-10978, 1992) instead of hsp90 and immobilized FK506 orFK506 analogs or hsp90. Binding to p23 can be assayed using recombinanthuman p23 (Johnson et al., Mol. Cell. Biol. 14:1956-1963, 1994) andimmobilized hsp90. Purified hsp70 and recombinant p60 (Dittmar et al.,J. Biol. Chem. 271:12833-12839, 1996) are also available for use in suchbinding assays.

Immunoassays can also be performed using conventional immunoassaymethodologies and antibodies that are specific for steroid receptorcomplex components, e.g., antibodies against FK2P52 (Tai et al.,Biochem. 25:5269-5275, 1986), hsp90 (Sanchez et al., J. Biol. Chem.260:12398-12401, 1985; Catelli et al., EMBO J. 4:3131-3135, 1985; Schuhet al., J. Biol. Chem. 260:14292-14296, 1985), hsp70 (a serum that alsorecognizes hsp90) (Erhart et al., Oncogene 3:595-603, 1988), p23(Johnson et al., Mol. Cell. Biol. 14:1956-1963, 1994), etc.

A well-accepted qualitative assay for receptor transformation, whichinvolves dissociation of hsp90 from the receptor complex, is conversionof a receptor complex to a state that binds polyanions such asphosphocellulose (Kalimi et al., J. Biol. Chem. 250:1080-1086, 1975;Atger and Milgrom, Biochem. 15:4298-4304, 1976), ATP-Sepharose (Toft etal., J. Steroid Biochem. 7:1053-1059, 1976; Miller and Toft, Biochem.17:173-177, 1978), and carboxymethol-Sephadex (Milgrom et al., Biochem.12:5198-5205, 1973; Parchman and Litwack, Arch. Biochem. Biophys.183:374-382, 1977).

An in vitro assay for nerve cell growth (neurite outgrowth) is providedin Example 1 below. In vivo assays for nerve regeneration are discussedin, for example, Gold et al., Restor. Neurol. Neurosci. 6:287-296, 1994;Gold et al., J. Neurosci. 15:7505-7516, 1995; Wang et al., J. Pharmacol.Exp. Therapeutics 282:1084-1093, 1997; Gold et al., Exp. Neurol.147:269-278, 1997; Gold et al., Soc. Neurosci. Abst. 23:1131, 1997,which examine the effects of systematic administration of a testcompound on nerve regeneration and functional recovery following a crushinjury to the rat sciatic nerve. Briefly stated, the right sciatic nerveof anaesthetized rats is exposed, and the nerve crushed twice usingforceps at the level of the hip. Following the sciatic nerve crush, thetest compound is administered to the rats, e.g., by subcutaneousinjection or oral administration. Functional recovery is assessed bydetermining the number of days following nerve crush until the animaldemonstrates onset of an ability to right its foot and move its toes,and the number of days until the animal demonstrates an ability to walkon its hind feet and toes. Nerve regeneration is also assessed bysampling tissues from the sciatic nerve at known (0.5 cm) distances fromthe crush site and examining the number of myelinated fibers and thesize of axons by light microscopy. The axons are also examined byelectron microscopy. Axonal areas of both myelinated and unmyelinatedfibers are determined by tracing the axolemma using a digitizing tabletconnected to a computer with appropriate software. Cumulative histogramsare constructed from these data and mean values and standard errors arecalculated to assess the effect of administration of the test compoundon axonal areas.

“Geldanamycin Derivatives”.

“Geldanamycin derivatives” include well-known synthetic derivatives(Schnur et al., J. Med. Chem. 38:3813-3820, 1995; Schnur et al., J. Med.Chem. 38:3806-3812, 1995). Geldanamycin derivatives preferably have thecarbamate group and ansa ring of geldanamycin, which are necessary foractivity (Schur et al., J. Med. Chem. 38:3806-3812, 1995), includingmodifications at the C23 methoxy and C22 methyl groups (Stebbins et al.,Cell 89:239-250, 1997). Geldanamycin derivatives are also discussed inU.S. Pat. Nos. 5,3877,584, 4,261,989, and 3,987,035, and in JapanesePatent Applications 88041885, 56100766, and 89002593, for example.

“FK506 Analogs”.

As used herein, the term “FK506 analogs” refers to compounds that arefunctionally analogous to FK506 in their ability to stimulate neuriticoutgrowth. Such FK506 analogs, such as V-10,367, retain the FKBP12binding domain but lack the structural components of the effector domain(FIG. 1) and may either bind FKBP12 or be non-binding. V-10,367, forexample, binds FKBP12 with high affinity (<1 nM) (Armistead et al., ActaCrystallogr. 51:522-528, 1995).

There has been an intense effort to design compounds that arestructurally related to FK506 and that share the ability of FK506 toinhibit FKBP12 and thereby cause immunosuppression. See, for example:Bierer et al., Science 250:556-559, 1990; Van Duyne et al., Science252:839-842, 1991; Van Duyne et al., J. Mol. Biol. 229:105-124, 1993;Hauske et al., J. Med. Chem. 35:4284-4296, 1992; Holt et al., J. Am.Chem. Soc. 115:9925-9938, 1993; Holt et al., Bioorg. Med. Chem. Lett.3:1977-1980, 1993; Teague and Stocks, Bioorg. Med. Chem. Lett.3:1947-1950, 1993; Wang et al., Bioorg. Med. Chem. Lett. 4:1161-1166,1994; Yamashita et al., Bioorg. Med. Chem. Lett. 4:325-328, 1994; Stockset al., Bioorg. Med. Chem. Lett. 4:1457-1460, 1994; Goulet et al.,Perspect. Drug Disc. Design 2:145-162, 1994; Wilson et al., Acta-Cryst.D51:511-521, 1995; Armistead et al., Acta Cryst. D51:522-528, 1995; U.S.Pat. Nos. 5,192,773, 5,330,993, 5,516,797, 5,612,350, 5,614,547,5,622,970, 5,654,332; and published international patent applications WO92/00278, WO 92/04370, WO 92/19593, WO 92/21313, WO 94/07858, and WO96/40633.

FK506 analogs include, but are not limited to:

(1) Compounds represented by the formula I (see U.S. Pat. Nos.5,622,970, 5,516,797, 5,330,993, 5,192,773, and WO 92/00278 regardingsynthesis of these compounds, the disclosures of which are incorporatedherein by reference):

wherein A is O, NH, or N—(C1-C4 alkyl);

wherein B is hydrogen, CHL—Ar, (C1-C6)-straight or branched alkyl,(C2-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl,(C5-C7)-cycloalkenyl or Ar substituted (C1-C6)-alkyl or (C2-C6)-alkenyl,or

wherein L and Q are independently hydrogen, (C1-C6)-straight or branchedalkyl or (C2-C6)-straight or branched alkenyl;

wherein T is Ar or substituted cyclohexyl with substituents at positions3 and 4 that are independently selected from the group consisting ofhydrogen, hydroxyl, O—(C1-C4)-alkyl or O—(C2-C4)-alkenyl and carbonyl;

wherein Ar is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyland phenyl having one to three substituents that are independentlyselected from the group consisting of hydrogen, halo, hydroxyl, nitro,CF₃, (C1-C6)-straight or branched alkyl or (C2-C6)-straight or branchedalkenyl, O—(C1-C4)- straight or branched alkyl or O—(C2-C4)-straight orbranched alkenyl, O-benzyl, O-phenyl, amino and phenyl;

wherein D is U; E is either oxygen or CH—Ur provided that if D ishydrogen, then E is CH—U or if E is oxygen, then D is not hydrogen;

wherein each U is independently selected from hydrogen,O—(C1-C4)-straight or branched alkyl or O—(C2- C4)-straight or branchedalkenyl, (C1-C6)-straight or branched alkyl or (C2-C6)-straight orbranched alkenyl, (C5-C7)-cycloalkyl or (C5-C7)-cycloalkenyl substitutedwith (C1-C4)-straight or branched alkyl or (C2-C4)- straight or branchedalkenyl, 2-indolyl, 3-indolyl, [(C1-C4)-alkyl or (C2-C4)-alkenyl]-Ar orAr;

wherein J is hydrogen or C1 or C2 alkyl or benzyl; K is (C1-C4)-straightor branched alkyl, benzyl or cyclohexylmethyl; or wherein J and K may betaken together to form a 5-7 membered heterocyclic ring that may containan oxygen (O), sulfur (S), SO or SO₂ substituent therein; and

the stereochemistry at position 1 is R or S.

(2) Compounds represented by the formula II (see U.S. Pat. No.5,654,332, WO 94/07858, and WO 92/19593 for synthesis of thesecompounds, the disclosures of which are incorporated herein byreference):

wherein A′ is CH₂, oxygen, NH, or N—(C1-C4 alkyl);

wherein B′ and W are independently hydrogen, Ar′, (C1-C10)-straight orbranched alkyl, (C2-C10)- straight or branched alkenyl or alkynyl,(C5-C7)- cycloalkyl substituted (C1-C6)-straight or branched alkyl,(C2-C6)-straight or branched alkenyl or alkynyl, (C5-C7)-cycloalkenylsubstituted (C1-C6)-straight or branched alkyl, (C2-C6)-straight orbranched alkenyl or alkynyl, or Ar′ substituted (C1-C6)-straight orbranched alkyl, (C2-C6)-straight or branched alkenyl or alkynyl whereinin each case, any one of the CH₂ groups of the alkyl, alkenyl, oralkynyl chains may be optionally replaced by a heteroatom selected fromthe group consisting of O, S, SO, SO₂, N, and NR, wherein R is selectedfrom the group consisting of hydrogen, (C1-C4)- straight or branchedalkyl, (C2-C4)-straight or branched alkenyl or alkynyl, and (C1-C4)bridging alkyl wherein a bridge is formed between the nitrogen and acarbon atom of the heteroatom-containing chain to form a ring, andwherein the ring is optionally fused to an Ar′ group, or

wherein Q′ is hydrogen, (C1-C6)-straight or branched alkyl or(C2-C6)-straight or branched alkenyl or alkynyl;

wherein T′ is Ar or substituted 5-7 membered cycloalkyl withsubstituents at positions 3 and 4 that are independently selected fromthe group consisting of oxo, hydrogen, hydroxyl, O—(C1-C4)-alkyl, andO—(C2-C4)- alkenyl;

wherein Ar′ is a carboxcyclic aromatic group selected from the groupconsisting of phenyl, 1-naphthyl, 2-naphthyl, indenyl, azulenyl,fluorenyl, and anthracenyl; or a heterocyclic aromatic group selectedfrom the group consisting of 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl,1,2,3-triazolyl, 1,3,4- thiadiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, 1,3,5- triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl, benzo[b]thiophenyl,lH-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, and phenoxazinyl;

wherein Ar′ may contain one to three substituents that are independentlyselected from the group consisting of hydrogen, halogen, hydroxyl,hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy,(C1-C6)-straight or branched alkyl, (C2-C6)-straight or branchedalkenyl, O—[(C1-C4)-straight or branched alkyl], O—[(C2-C4)-straight orbranched alkenyl], O-benzyl, O- phenyl, 1,2-methylenedioxy, amino,carboxyl, N-[(C1-C5)- straight or branched alkyl or (C2-C5)-straight orbranched alkenyl)carboxamides, N,N-di[(C1-C5)-straight or branched alkylor (C2-C5)-straight or branched alkenyl]carboxamides,N-morpholinocarboxamide, N- benzylcarboxamide,N-thiomorpholinocarboxamide, N- picolinoylcarboxamide, O—X,CH₂—(CH₂)_(q)—X, O—(CH₂)_(q)—X, (CH₂)_(q)—O—X, and CH═CH—X;

wherein X is 4-methoxyphenyl, 2-pyridyl, 3- pyridyl, 4-pyridyl, pyrazyl,quinolyl, 3,5- dimethylisoxazoyl, isoxazoyl, 2-methylthiazoyl, thiazoyl,2-thienyl, 3-thienyl, or pyrimidyl; and q is 0-2;

wherein G is U′;

wherein M is either oxygen or CH—U′; provided that if G is hydrogen,then M is CH—U′ or if M is oxygen, then G is U′;

wherein U′ is hydrogen, O—[(C1-C4)-straight or branched alkyl] orO—[(C2-C4)-straight or branched alkenyl], (C1-C6)-straight or branchedalkyl or (C2-C6)- straight or branched alkenyl, (C5-C7)-cycloalkyl or(C5- C7)-cycloalkenyl substituted with (C1-C4)-straight or branchedalkyl or (C2-C4)-straight or branched alkenyl, [(C1-C4)-alkyl or(C2-C4)-alkenyl]-Y or Y;

wherein Y is selected from the group consisting of phenyl, 1-naphthyl,2-naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl, 2-pyrrolinyl,3-pyrrolinyl, pyrolidinyl, 1,3-dioxolyl, 2-imidazolinyl, imidazolidinyl,2H-pyranyl, 4H-pyranyl, piperidyl, 1,4- dioxanyl, morpholinyl,1,4-dithianyl, thiomorpholinyl, piperazinyl, quinuclidinyl, andheterocyclic aromatic groups as defined for Ar′ above;

wherein Y may contain one to three substituents that are independentlyselected from the group consisting of hydrogen, halogen, hydroxyl,hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy,(C1-C6)-straight or branched alkyl, (C2-C6)-straight or branchedalkenyl, O—[(C1-C4)-straight or branched alkyl], O—[(C2-C4)-straight orbranched alkenyl], O-benzyl, O-phenyl, 1,2- methylenedioxy, amino, andcarboxyl;

wherein J′ is hydrogen, (C1-C2) alkyl or benzyl; wherein K is(C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl, orwherein J′ and K may be taken together to form a 5-7 memberedheterocyclic ring that may contain a heteroatom selected from the groupconsisting of O, S, SO and SO₂;

wherein m is 0-3; and

wherein the stereochemistry at position 1 is R or S and thestereochemistry at position 2 is R or S.

(3) Compounds represented by the formula III (see Armistead et al., ActaCryst. D51:522-528, 1995, including a discussion of selection of R andof the synthesis of these compounds, the disclosure of which isincorporated herein by reference):

(4) Compounds represented by the formula IV (see WO 92/21313, includinga discussion of the synthesis of these compounds, the disclosure ofwhich is incorporated herein by reference):

wherein A is CH₂, oxygen, NH or N—(C1-C4 alkyl);

wherein B and D are independently Ar, hydrogen, (C1-C6)-straight orbranched alkyl, (C1-C6)-straight or branched alkenyl, (C1-C6)-straightor branched alkyl or alkenyl that is substituted with a(C5-C7)-cycloalkyl, (C1-C6)-straight or branched alkyl or alkenyl thatis substituted with a (C5-C7)-cycloalkenyl, or Ar substituted(C1-C6)-straight or branched alkyl or alkenyl, wherein, in each case,one or two of the CH₂ groups of the alkyl or alkenyl chains may contain1-2 heteroatoms selected from the group consisting of oxygen, sulfur, SOand SO₂ in chemically reasonable substitution patterns, or:

provided that both B and D are not hydrogen;

wherein Q is hydrogen, (C1-C6)-straight or branched alkyl or(C1-C6)-straight or branched alkenyl;

wherein T is Ar or substituted 5-7 membered cycloalkyl with substituentsat positions 3 and 4 that are independently selected from the groupconsisting of hydrogen, hydroxyl, O—(C1-C4)-alkyl, O—(C1-C4)-alkenyl andcarbonyl;

wherein Ar is selected from the group consisting of phenyl, 1-naphthyl,2-naphthyl, 2-furyl, 3- furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4- pyridyl, monocyclic and bicyclic heterocyclic ring systemswith individual ring sizes being 5 or 6 that may contain in either orboth rings a total of 1-4 heteroatoms independently selected from O, Nand S; wherein Ar may contain one to three substituents that areindependently selected from the group consisting of hydrogen, halo,hydroxyl, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-straight orbranched alkyl, (C2-C6)-straight or branched alkenyl, O—(C1-C4)-straightor branched alkyl, O—(C2-C4)-straight or branched alkenyl, O-benzyl,O-phenyl, 1,2-methylenedioxy, amino, carboxyl and phenyl;

wherein E is (C1-C6)-straight or branched alkyl, (C1-C6)-straight orbranched alkenyl, (C5-C7)- cycloalkyl, (C5-C7)-cycloalkenyl substitutedwith (C1-C4)-straight or branched alkyl or (C1-C4)-straight or branchedalkenyl, [(C2-C4)alkyl or (C2-C4)-alkenyl)]-Ar or Ar (Ar as describedabove);

wherein J is hydrogen or Cl or C2 alkyl or benzyl; K is (C1-C4)-straightor branched alkyl, benzyl or cyclohexylmethyl; or wherein J and K may betaken together to form a 5-7 membered heterocyclic ring that may containan oxygen, sulfur, SO or SO₂ substituent therein; and;

wherein n is 0-3; and

wherein the stereochemistry at position 1 is R or S and thestereochemistry at position 2 being R or S.

(5) Compounds represented by the formula V (see WO 92/04370, including adiscussion of the synthesis of these compounds, the disclosure of whichis incorporated herein by reference):

wherein A is NH, O, S, or CH;

wherein if A is NH, O, or S, B is PCO— or POCO—, where P is a C1-C6straight or branched alkyl or alkenyl group, a C5-C6 cycloalkyl orcycloalkenyl, or a methyl substituted with a C5-C6 cycloalkyl, C5-C6cycloalkenyl, phenyl, 1-naphthyl, 2-naphthyl, 9- fluorenyl, or1-adamantyl;

wherein if A is CH, then B is connected via a trans double bond and is aC2-C4 straight or branched alkyl or alkenyl group, or is a methyl orethyl substituted with either a C5-C6 cyclic alkyl group or Ar, where Aris selected from the group consisting of 1- naphthyl, 2-naphthyl,2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one to threesubstituents that are independently selected from the group consistingof: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branched alkyl oralkenyl, O—(C1-C4) straight or branched alkyl or alkenyl, and Ar, whereAr is selected from the group consisting of 1-naphthyl, 2-naphthyl,2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one to threesubstituents that are independently selected from the group consistingof: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branched alkyl oralkenyl, O—(C1-C4) straight or branched alkyl or alkenyl; wherein nomore than two Ar groups may be linked together;

wherein D is hydrogen, C1-C4 straight or branched alkyl or alkenyl,hydroxy, tert-butyloxy, benzyloxy, 4-benzyloxyphenyl, cyclohexyl,—(CH₂)_(n)—CO₂—Q, where n=0 or 1 and Q is methyl, ethyl, i-propyl,t-butyl, benzyl, 1-naphthyl, 2-naphthyl, or cyclohexyl; or Ar, where Aris selected from the group consisting of 1- naphthyl, 2-naphthyl,2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one to threesubstituents that are independently selected from the group consistingof: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branched alkyl oralkenyl, O—(C1-C4) straight or branched alkyl or alkenyl, and Ar, whereAr is selected from the group consisting of 1-naphthyl, 2-naphthyl,2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one to threesubstituents that are independently selected from the group consistingof: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branched alkyl oralkenyl, O—(C1-C4) straight or branched alkyl or alkenyl; wherein nomore than two Ar groups may be linked together;

wherein E and K are independently hydrogen or methyl;

wherein G is either methyl or ethyl; J is hydrogen, C1-C6 straight orbranched alkyl or alkenyl, C6-C6 cycloalkyl or cycloalkenyl, sulfhydryl,hydroxy, phenyl, 3-indolyl, or benzyl; wherein G and J may be connectedby a bond to form a cycle of 5 or 6 members;

wherein L is O or an α-amino acid residue attached via the α-nitrogen,and selected from the group consisting of: alanine, 2-aminobutyric acid,valine, norvaline, leucine, norleucine, isoleucine, phenylalanine,cyclohexylalanine, tryptophan, 1- naphthylalanine, 2-naphthylalanine,threonine (side chain benzyl or tert-butyl ether), methionine, or serine(side chain benzyl or tert-butyl ether);

wherein if L is O, then M is C1-C6 straight or branched alkyl oralkenyl, or —(CH₂)_(n)—Ar, where n=1-6 and Ar is selected from the groupconsisting of: 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl,phenyl and phenyl having one to three substituents that areindependently selected from the group consisting of: hydroxyl, halo,nitro, CF₃, C1-C4 straight or branched alkyl or alkenyl, O—(C1-C4)straight or branched alkyl or alkenyl, and Ar, wherein Ar is selectedfrom the group consisting of: 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl,2-thienyl, phenyl and phenyl having one to three substituents that areindependently selected from the group consisting of: hydroxyl, halo,nitro, CF₃, C1-C4 straight or branched alkyl or alkenyl, O—(C1-C4)straight or branched alkyl or alkenyl; wherein no more than two Argroups may be linked together;

wherein if L is an amino acid, then M is O—(C1-C4) straight or branchedalkyl, O-benzyl, NH-Phenyl, or NH-4-nitrophenyl and is attached to theamino acid carbonyl;

the stereochemistry at all positions being R or S, and preferably thestereochemistry is S at L if L is an α-amino acid, and at thosepositions marked with asterisks; however, when J is sulfhydryl, thepreferred stereochemistry of the asterisked position immediatelyadjacent to the nitrogen is R.

(6) Compounds represented by the formula VI (see WO 96/40633, includinga discussion of the synthesis of these compounds, the disclosure ofwhich is incorporated herein by reference):

wherein, R1 is selected from the group consisting of a C1-C9 straight orbranched chain alkyl or alkenyl group optionally substituted with C3-C8cycloalkyl, C3 or C5 cycloalkyl, C5-C7 cycloalkenyl, or Ar1, where saidalkyl, alkenyl, cycloalkyl or cycloalkenyl groups may be optionallysubstituted with C1-C4 alkyl, C1-C4 alkenyl, or hydroxy, where Ar1 isselected from the group consisting of 1-naphthyl, 2- naphthyl,2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2- thiazolyl, 2-thienyl,3-thienyl, 2-, 3-, 4-pyridyl, and phenyl, having one to threesubstituents that are independently selected from the group consistingof hydrogen, halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight orbranched alkyl or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy, phenoxy,benzyloxy, and amino;

wherein X is selected from the group consisting of oxygen, sulfur,methylene (CH₂ ), or H₂ ;

wherein Y is selected from the group consisting of oxygen or NR₂, whereR₂ is hydrogen or C1-C6 alkyl; and

wherein Z is selected from the group consisting of C2-C6 straight orbranched chain alkyl or alkenyl, wherein the alkyl chain is substitutedin one or more positions with Ar₁ as defined above, C3-C8 cycloalkyl,cycloalkyl connected by a C1-C6 straight or unbranched alkyl or alkenylchain and Ar₂ 1where Ar₂ is selected from the group consisting of2-indolyl, 3-indolyl, 2-furyl, 3- furyl, 2-thiazolyl, 2-thienyl,3-thienyl, 2-, 3-, or 4- pyridyl, and phenyl, having one to threesubstituents that are independently selected from the group consistingof hydrogen, halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight orbranched alkyl or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy, phenoxy,benzyloxy, and amino;

wherein Z may also be the fragment:

where

R₃ is selected from the group consisting of straight or branched alkylC1-C8 optionally substituted with C3-C8 cycloalkyl, or Ar, as definedabove, and unsubstituted Ar₁;

X₂ is O or NR₅ where R₅ is selected from the group consisting ofhydrogen, C1-C6 straight or branched alkyl and alkenyl;

R₄ is selected from the group consisting of phenyl, benzyl, C1-C5straight or branched alkyl or alkenyl, and C1-C5 straight or branchedalkyl or alkenyl substituted with phenyl;

wherein the stereochemistry at position 1 is R or S.

Also encompassed are pharmaceutically acceptable derivatives of theFK506 analogs, including, but not limited to, any pharmaceuticallyacceptable salt, ester, salt of an ester, or any other derivative which,upon administration to a patient, is capable of providing directly orindirectly a non-binding FK506 analog or a metabolite or residue thereofthat has the desired neurotrophic activity. Included within the scope ofthe invention are enantiomers, the racemic form, and diastereoisomericmixtures. Enantiomers and diastereoisomers can be separated byconventional methods.

Formulae I-VI above represent compounds that have a wide range ofbinding affinities for FKBP12. The mechanism for neurotrophic activityof FK506 presented herein indicates that the effectiveness of FK506 andFK506 analogs in stimulating nerve cell growth is unrelated to theirability to bind FKBP12. Instead, their effectiveness in stimulatingnerve cell growth relates to ability of such compounds to bind FKBP52and subsequently interfere with the interaction of FKBP52 and hsp90 in asteroid receptor complex, e.g., by competing for FKBP52 binding tohsp90, altering the conformation of FKBP52, etc.

A “non-binding FK506 analog” is defined as an FK506 analog that does notbind to FKBP12. Preferably, such FK506 analogs bind FKBP12 with anapparent K_(d) of greater than 10 μM as measured using well-knownassays, and preferably greater than 30 μM, and more preferably greaterthan 100 μM. Values for the apparent K_(d) can be determined, forexample, by a competitive LH-20 binding assay performed as described,for example, in Harding et al., Nature 341:758-760, 1989 (using 32-[1-¹⁴C]-benzoyl FK506 as a reporting ligand; Siekierka et al., Nature341:755-757, 1989, using [³H]dihydro-FK506 as a reporting ligand); andU.S. Pat. No. 5,654,332.

Alternatively, a “non-binding FK506 analog” is defined as an FK506analog that does not significantly inhibit FKBP12 rotomase activity whenadministered to a patient at dosage levels of about 0.01 to about 100mg/kg body weight/day. Assays for inhibition of FKBP12 rotamase activityare described in Harding et al. (Nature 341:758-760, 1989), Siekierka etal., Nature 341:755-757, 1989, and U.S. Pat. No. 5,654,332, for example.The assays of Harding et al. and Siekierka et al. employ a reactionmixture that includes the cis form of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, FKBP12, a test compound, andchymotrypsin, and spectrophotometrically measure the release ofp-nitroanilide as a result of isomerization of the substrate.

Non-binding FK506 analogs are non- immunosuppressive, as can bedemonstrated by well-known assays, e.g., as discussed in U.S. Pat. No.5,516,797, WO 92/21313, WO 92/19593, and WO 92/04370.

Non-binding FK506 analogs can be used in the form of salts preferablyderived from inorganic or organic acids and bases, including, but notlimited to:

acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3- phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base saltsinclude, but are not limited to, ammonium salts, alkali metal salts(such as sodium and potassium salts), alkaline earth metal salts (suchas calcium and magnesium salts), salts with organic bases (such asdicyclohexylamine salts), N-methyl-D-glucamine, and salts with aminoacids (such as arginine, lysine, etc.). Basic nitrogen-containing groupscan be quaternized, e.g., with such agents as lower alkyl halides (suchas methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides),dialkyl sulfates (such as dimethyl, diethyl, dibutyl, an diamylsulfates), long- chain halides (such as decyl, lauryl, myristyl, andstearyl chlorides, bromides, and iodides), aralkyl halides (such asbenzyl and phenethyl bromides), etc. Water or oil-soluble or dispersibleproducts are produced thereby.

Non-binding FK506 analogs can be modified by appending appropriatefunctionalities by well-known methods to enhance selected biologicalproperties, including increasing penetration of the analogs into a givencellular compartment (e.g., blood, lymphatic system, central nervoussystem, etc.), increase oral availability, increase solubility to permitadministration by injection, alter metabolism, and alter rate ofexcretion, for example.

Preferably, the non-binding FK506 analogs have a molecular weight belowabout 750 atomic mass units (a.m.u.) (as the parent compound, althoughthe salts of such compounds can have higher molecular weights).

“Effective Amount” or “Nerve Growth Stimulating Amount.”

An “effective amount” or a “nerve growth stimulating amount” of acomposition according to the invention is an amount sufficient toachieve a statistically significant promotion of nerve cell growth orregeneration compared to a control. Nerve cell growth or nerveregeneration can be readily assessed using an in vitro assay, e.g., theassay described in the Examples below. Alternatively, nerve cell growthor regeneration can be determined in an in vivo assay or by direct orindirect signs of nerve cell growth and regeneration in a patient.Preferably, the increase in nerve cell growth or regeneration is atleast 10%, preferably at least 30%, and most preferably 50% or morecompared to a control. Preferred dosage levels are between about 0.1 toabout 400 mg/kg per day of the FK506 analog for subcutaneous delivery.For oral administration, preferred dosage levels are between about 0.01to about 40 mg/kg/day.

Therapeutic and Prophylactic Uses

Pharmaceutical compositions according to the invention can beperiodically administered to a mammalian patient (e.g., a humanpatient), in need of such treatment, to promote neuronal regenerationand functional recovery and to stimulate neurite outgrowth and therebyto treat various neuropathological states, including damage toperipheral nerves and the central nervous system caused by physicalinjury (e.g., spinal cord injury and trauma, sciatic or facial nervelesion or injury), disease (e.g., diabetic neuropathy), cancerchemotherapy (e.g., by vinca alkaloids and doxorubicin), brain damageassociated with stroke and ischemia associated with stroke, andneurological disorders including, but not limited to, various peripheralneuropathic and neurological disorders related to neurodegenerationincluding, but not limited to: trigeminal neuralgia, glossopharyngealneuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy,amyotrophic lateral sclerosis, progressive muscular atrophy, progressivebulbar inherited muscular atrophy, herniated, ruptured or prolapsedvertebral disk syndromes, cervical spondylosis, plexus disorders,thoracic outlet destruction syndromes, peripheral neuropathies such asthose caused by lead, acrylamides, gamma-diketones (glue- sniffer'sneuropathy), carbon disulfide, dapsone, ticks, porphyria, Gullain-Barresyndrome, Alzheimer's disease, Parkinson's disease, and Huntington'schorea.

In addition, pharmaceutical compositions according to the presentinvention display a wide range of other therapeutic or prophylacticproperties, including, treatment of stroke (see, e.g., Sharkey andButcher, Nature 371:336-339, 1994, Vagita et al., Life Sciences59:1643-1650, 1996; Tokime et al., Neurosci. Lett. 206:81-84, 1996;Drake et al., Acta. Physiol. Scand. 158:155-159, 1996; and Kuroda etal., Neurosci. Res. Comm. 19:83-90, 1996), AIDS dementia (see, e.g.,Dawson and Dawson, Adv. Neuroimmunol. 4:167-173, 1994; and Sekigawa etal., J. Clin. Immunol. 15:312-317, 1995); hair growth (Yamamoto et al.,J. Investig. Dermatol. 102:160-164, 1994; Jiang et al., J. Investig.Dermatol. 104:523-525, 1995); and connective tissue disorders (see e.g.,Steinmann et al., J. Biol. Chem. 266:1299-1303, 1991), and as a malecontraceptive (see e.g., Hisatomi et al., Toxicology 109:75-83, 1996).

A transection of a periphal nerve or a spinal cord injury can be treatedby administering a nerve growth stimulating amount of anon-FKBP12-binding nerve growth promoting agent to the mammal andgrafting to the peripheral nerve or spinal cord an allograft (Osawa etal., J. Neurocytol. 19:833-849, 1990; Buttemeyer et al., Ann. PlasticSurgery 35:396-401, 1995) or an artificial nerve graft (Madison andArchibald, Exp. Neurol. 128:266-275, 1994; Wells et al., Exp. Neurol.146:395-402, 1997). The space between the transected ends of theperipheral nerve or spinal cord is preferably filled with a non-cellular gap-filling material such as collagen, methyl cellulose, etc.,or cell suspensions that promote nerve cell growth, such as Schwanncells (Xu et al., J. Neurocytol. 26:1-16, 1997), olfactory cells, andsheathing cells (Li et al. Science 277:2000-2002, 1997). The nervegrowth promoting agent can be included together with with such cellularor non-cellular gap-filling materials.

Pharmaceutical Formulations

Pharmaceutical formulations according to the present invention encompassformulations comprising (1) an amount (for example, a unit dosage) of anNGPA together with (2) one or more well-known non-toxic pharmaceuticallyacceptable excipients, including carriers, diluents, and/or adjuvants,and optionally (3) one or more biologically active ingredients. Standardpharmaceutical formulation techniques are used, such as those disclosedin Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(latest edition).

A pharmaceutical formulation according to the invention includes one ormore NGPAs and can also include, for example, one or more otherbiologically active ingredients, including, but not limited to FK506 oran FKBP12-binding FK506 analogs or one or more other neurotrophicagents, including, for example, NGF, IGF-1, aFGF, bFGF, PDGF, BDNF,CNTF, GDNF, NT-3, and NT 4/5; and so on.

It is preferred that the pharmaceutical formulation includes an amountof a neurotrophic agent(s), preferably NGF, such that the patientreceives a dosage of between about 0.01 to 100 μg/kg body weight/day ofthe neurotrophic agent, or that the neurotrophic agent be administeredseparately, e.g., in separate single or multiple dosage forms,preferably concurrently, consecutively, or within less than about fivehours of each other.

The compositions can be in the form of tablets, capsules, powders,granules, lozenges, liquid or gel preparations, such as oral, topical,or sterile parenteral solutions or suspensions (e.g., eye or ear drops,throat or nasal sprays, etc.), transdermal patches, and other formsknown in the art.

Such pharmaceutical compositions can be administered systemically orlocally in any manner appropriate to the treatment of a given condition,including orally, parenterally, rectally, nasally, buccally, vaginally,topically, optically, by inhalation spray, or via an implantedreservoir. The term “parenterally” as used herein includes, but is notlimited to subcutaneous, intravenous, intramuscular, intrasternal,intrasynovial, intrathecal, intrahepatic, intralesional, andintracranial administration, for example, by injection or infusion. Fortreatment of the central nervous system, the pharmaceutical compositionspreferably readily penetrate the blood-brain barrier when peripherallyadministered or are administered intraventricularly.

Pharmaceutically acceptable carriers include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, serum proteins(such as human serum albumin), buffers (such as phosphates), glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat, for example.

Tablets and capsules for oral administration can be in a form suitablefor unit dose presentation and can contain conventional pharmaceuticallyacceptable excipients. Examples of these include binding agents such assyrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone;fillers such as lactose, sugar, corn starch, calcium phosphate,sorbitol, or glycine; tableting lubricants, such as magnesium stearate,talc, polyethylene glycol, or silica; disintegrants, such as potatostarch; and dispersing or wetting agents, such as sodium lauryl sulfate.The tablets can be coated according to methods well known in normalpharmaceutical practice. Oral liquid preparations can be in the form of,for example, aqueous or oily suspensions, solutions, emulsions, syrupsor elixirs, or can be presented as a dry product for reconstitution withwater or other suitable vehicle before use. Such liquid preparations cancontain conventional additives such as suspending agents, e.g.,sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenatededible fats, emulsifying agents, e.g., lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (including edible oils), e.g., almond oil,fractionated coconut oil, oily esters such as glycerine, propyleneglycol, or ethyl alcohol; preservatives such as methyl or propylp-hydroxybenzoate or sorbic acid, and, if desired, conventionalflavoring or coloring agents.

Pharmaceutical compositions according to the present invention can alsobe administered parenterally in a sterile aqueous or oleaginous medium.The composition can be dissolved or suspended in a non-toxicparenterally-acceptable diluent or solvent, e.g., as a solution in1,3-butanediol. Adjuvants such as local anesthetics, preservatives, andbuffering agents can also be dissolved in the vehicle. Commonly usedvehicles and solvents include water, physiological saline, Hank'ssolution, Ringer's solution, and sterile, fixed oils, includingsynthetic mono- or di-glycerides, etc. Fatty acids, such as oleic acidand its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as Ph. Helv or a similar alcohol.

For topical application, the drug may be made up into a solution,suspension, cream, lotion, ointment in a suitable aqueous or non-aqueousvehicle. Additives may also be included, e.g., buffers such as sodiummetabisulphite or disodium edeate; preservatives such as bactericidaland fungicidal agents, including phenyl mercuric acetate or nitrate,benzalkonium chloride or chlorhexidine, and thickening agents, such ashypromellose.

The dosage unit involved depends, for example, on the condition treated,nature of the formulation, nature of the condition, embodiment of theclaimed pharmaceutical compositions, mode of administration, andcondition and weight of the patient. Dosage levels on the order of about0.1 to about 400 mg/kg per day of the active ingredient are useful inthe treatment of the conditions listed above.

The invention will be better understood by reference to the followingexamples, which are intended to merely illustrate the best mode nowknown for practicing the invention. The scope of the invention is not tobe considered limited thereto, however.

EXAMPLES

FK506 and Geldanamycin Promote Nerve Regeneration by a Common Mechanism

Materials and Methods

Cell Cultures.

SH-SY5Y human neuroblastoma cells were maintained in DMEM medium (GIBCO)supplemented with 10% fetal calf serum (SIGMA), 50 IU/mL penicillin, and50 mg/mL streptomycin (GIBCO) at 37° C. in 7% CO². Cells were plated insix-well plates at 1×10⁶ cells/well and treated with 0.4 mM aphidicolin(SIGMA). At five days, cells were washed, treated with nerve growthfactor (NGF) (Boehringer Mannheim, Indianapolis, Ind.) at 10 ng/mL (toinduce process outgrowth) in the presence or absence of FK506 (1 and 10nM) (Calbiochem-Novabiochem Int'l., La Jolla, Calif.) and/orgeldanamycin (0.1, 1, and 10 nM) (Calbiochem-Novabiochem, La Jolla,Calif.). Media was changed at 96 hours and replaced with fresh mediawith the compounds (NGF plus FK506 and/or geldanamycin) for anadditional 72 hours (total time, 168 hours). The top 50% of axonallengths were selected for statistical analysis. All experiments were runin duplicate wells and repeated at least twice for reproducibility.

Light Morphometry of Neurite Lengths.

For analysis of process length, cells 20 fields per well) were randomlyphotographed at 72 and 168 hours. Neurite lengths were measured onphotographic prints using a Houston Instrument HI-PAD digitizing tabletconnected to an IBM XT computer with appropriate software (Bioquant IV,R&M Biometrics, Nashville, Tenn.); only those processes greater than twotimes the cell body length were measured. Data from identically treatedwells were not different and were therefore combined. Mean values andhistograms were constructed from these data. Histograms were comparedusing a Mann-Whitney U test, which makes no assumptions about the shapeof the distribution.

Preparation of FK506 and Geldanamycin. FK506 (mol. wt. 822) andgeldanamycin (mol. wt. 561) were dissolved in DMEM medium.

Results

In a first set of experiments, SH-SY5Y neuroblastoma cells were platedin 6-well plates with DMEM plus 15% FCS and differentiated with NGF (10ng/ml). The effects of various concentrations of geldanamycin and FK506on neurite growth, alone and in combination, were tested. The meanlengths of neuritic processes of untreated and treated cells are shownin Table 1.

The cells developed long axonal-like processes upon exposure to NGF (10ng/mL) as measured at 168 hours after treatment. NGF more than doubledthe mean length of the processes compared to untreated cells (compareFIGS. 1 and 2). An even greater increase in the length of the processeswas observed when the cells were exposed to geldanamycin at 1 nM in thepresence of NGF (FIG. 3). However, geldanamycin at 10 nM had no effect(FIG. 4). FK506 (10 nM) stimulated neurite outgrowth in the presence ofNGF (FIG. 5) to a greater degree than geldanamycin at either 1 nM or 10nM. In combination with FK506 (10 nM), geldanamycin (FIG. 6, 1 nM; FIG.7, 10 nM) inhibited the effect of FK506 in a concentration- dependentfashion.

In a second set of experiments, lower concentrations of geldanamycin andFK506 were tested, alone and in combination. The mean lengths ofneuritic processes of untreated and treated cells are shown in Table 2.NGF-treated cells (FIG. 9) had mean neuritic lengths more than doublethe mean length of untreated control cells (FIG. 8). Nerve growthstimulation was observed with FK506 (1 nM, FIG. 10; 10 nM, FIG. 11) inthe presence of NGF and geldanamycin (0.1 nM, FIG. 12) in the presenceof NGF. The lower concentration of FK506 (1 nM) was more effective instimulating neurite outgrowth than the higher concentration (10 nM), andgeldanamycin at 0.1 nM was even more effective in stimulating neuriteoutgrowth than FK506 at either 1 nM or 10 nM. Combined treatment withgeldanamycin (0.1 nM) and FK506 (1 nM, FIG. 13; 10 nM, FIG. 14) in thepresence of NGF showed that the effects of geldanamycin and FK506 wereadditive, particularly at the lower FK506 concentration (FIG. 13).

Geldanamycyin and FK506 each stimulate neurite outgrowth in aconcentration-dependent fashion. Taken together, the similar effects ofgeldanamycin and FK506 in stimulating neurite outgrowth, their additiveeffects at low concentrations, and their inhibitory effects at highconcentrations (like high concentrations of either compound alone),demonstrate that the two compounds act on nerve cells via a commonmechanism. That mechanism likely involves an interaction of bothcompounds with components of steroid receptor complexes. FKBP12 does notappear to play a role in the stimulation of neurite outgrowth by eithergeldanamycin or FK506.

In further experiments, we have found that estrogen (10 nM) anddexamethasone (10 nM) increased neurite outgrowth (without NGF) ofSH-SY5Y cells and produced an additive effect on neurite outgrowth(neurotrophic action) with FK506 (10 nM).

This invention has been detailed both by example and by directdescription. It should be apparent that one having ordinary skill in therelevant art would be able to surmise equivalents to the invention asdescribed in the claims which follow but which would be within thespirit of the foregoing description. Those equivalents are to beincluded within the scope of this invention.

TABLE 1 Mean Length of Top 50% of Neuritic Processes of SH-SY5Y 168Hours After Treatment with Geldanamycin (1 nM or 10 nM) and/or FK506 (10nM) in the Presence of NGF Treatment Mean Length (μM) S.E.M. Untreated41.61 1.25 NGF (10 ng/mL) 53.99 2.26 Geldanamycin (1 nM) + 64.00 2.36NGF (10 ng/mL) Geldanamycin (10 nM) + 54.81 2.10 NGF (10 ng/mL) FK506(10 nM) + 77.82 2.70 NGF (10 ng/mL) Geldanamycin (1 nM) + 72.63 2.01FK506 (10 nM) + NGF (10 ng/mL) Geldanamycin (10 nM) + 67.41 1.67 FK506(10 nM) + NGF (10 ng/mL)

TABLE 2 Mean Length of Top 50% of Neuritic Processes of SH-SY5Y 168Hours After Treatment with Geldanamycin (0.1 nM) and/or FK506 (1 nM or10 nM) in the Presence of NGF Treatment Mean Length (μM) S.E.M.Untreated 31.86 1.56 NGF (10 ng/mL) 70.38 6.61 Geldanamycin (0.1 nM) +98.07 5.72 NGF (10 ng/mL) FK506 (1 nM) + 89.92 6.40 NGF (10 ng/mL) FK506(10 nM) + 82.68 5.22 NGF (10 ng/mL) Geldanamycin (0.1 nM) + 110.51  6.13FK506 (1 nM) + NGF (10 ng/mL) Geldanamycin (0.1 nM) + 92.50 6.40 FK506(10 nM) + NGF (10 ng/mL)

2 1 8 PRT Sylvilagus sp. 1 Glu Asp Leu Thr Asp Asp Glu Asp 1 5 2 8 PRTRattus sp. 2 Arg Lys Thr Lys Lys Lys Ile Lys 1 5

What is claimed is:
 1. A method of identifying a compound thatstimulates nerve cell growth, comprising: assaying a plurality of testcompounds for binding to a polypeptide component of a steroid receptorcomplex other than a steroid hormone binding portion of the complexassaying the compound for inhibiting assembly of the complex or forpromoting dissociation of the complex; and assaying the compound forstimulating nerve cell growth.
 2. The method of claim 1, wherein thepolypeptide component of the steroid receptor complex is hsp90.
 3. Themethod of claim 1, wherein the plurality of test compounds are selectedfrom the group consisting of a non- FKBP12-binding FK506 analog, abenzoquinone ansamycin, a peptide comprising a sequence of a selectedpolypeptide component of the steroid receptor complex at a site ofinteraction between the selected component and another polypeptidecomponent of the steroid receptor complex, an antibody that binds to apolypeptide component of the complex, and combinations thereof.
 4. Themethod of claim 3, wherein the test compound is a benzoquinoneansamycin.
 5. The method of claim 4, wherein the test compound is ageldanamycin derivative.
 6. The method of claim 1, wherein assaying aplurality of test compounds for binding to a polypeptide component ofthe steroid receptor complex comprises identifying test compounds thatbind to FKBP12 with an apparent K_(d) of greater than 10 μM.
 7. Themethod of claim 6, wherein the test compound binds FKBP12 with anapparent Kd of greater than 30 μM.
 8. The method of claim 7, wherein thetest compound binds FKBP12 with an apparent Kd of greater than 100 μM.9. The method of claim 1, wherein the test compounds are selected fromthe group consisting of a peptide comprising a sequence from a componentof the steroid receptor complex which peptide inhibits assembly orpromotes dissociation of the steroid receptor complex, an antibody thatbinds to a component of the steroid receptor complex and preventsassembly or promotes dissociation of the steroid receptor complex, abenzoquinone ansamycin, and a FK506 analog which binds to FKBP12 with aK_(d) of at least 10 μM, and combinations thereof.
 10. The method ofclaim 1, wherein the polypeptide component of a steroid receptor complexis FKBP52.
 11. The method of claim 2, wherein the plurality of compoundscomprises compounds selected from the group consisting of a FK506analog, a benzoquinone ansamycin, a peptide comprising a sequence of aselected polypeptide component of the steroid receptor complex at a siteof interaction between the selected component and another polypeptidecomponent of the steroid receptor complex, an antibody that binds to apolypeptide component of the complex, and combinations thereof.
 12. Themethod of claim 10, wherein the plurality of compounds comprises ageldanamycin or a derivative thereof.
 13. The method of claim 10,wherein the plurality of compounds comprises compounds selected from thegroup consisting of a FK506 analog, a benzoquinone ansamycin, a peptidecomprising a sequence of a selected polypeptide component of the steroidreceptor complex at a site of interaction between the selected componentand another polypeptide component of the steroid receptor complex, anantibody that binds to a polypeptide component of the complex, andcombinations thereof.
 14. The method of claim 1, wherein the compound isa FK506 analog.
 15. The method of claim 1, wherein the compound is abenzoquinone ansamycin.
 16. The method of claim 1, wherein the compoundis a peptide comprising a sequence of a selected polypeptide componentof the steroid receptor complex at a site of interaction between theselected component and another polypeptide component of the steroidreceptor complex.
 17. The method of claim 1, wherein the compound is anantibody that binds to a polypeptide component of the complex.